This research will examine the influence of previous exposure to a mixture of heavy metals (Cd, Cu, Pb, Zn) on population and community responses to subsequent stress. We will evaluate mechanisms responsible for enhanced tolerance by relating responses at the molecular and population level to change observed at higher levels of organization. Studies will be conducted using benthic macroinvertebrate populations and communities collected from the Arkansas River, a U.S. EPA Superfund site that has been polluted by metals for over 100 years. At the population level, we hypothesize that tolerance of macroinvertebrates to metals results from increased production of metal-binding proteins. Studies conducted in collaboration with Dr. Barry Beaty (Project 5) will investigate molecular changes associated with increased production of metal-binding proteins. We hypothesize that increased tolerance at the population level will have cascading effects on higher levels of organization. At the community level. We will assess the relative importance of both intraspecific (physiological and/or genetic changes) and interspecific (replacement of sensitive species by tolerant species) tolerance mechanisms. Finally, this research will investigate the effects of novel stressors (e.g., increased temperature, acidification) on previously-exposed and naive systems. Because increased tolerance to metals may be associated with decreased genetic heterogeneity and increased metabolic costs, we hypothesize that systems previously-exposed to metals will be more susceptible to novel stressors.